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Background The pathogenesis of beryllium-induced pulmonary fibrosis is unknown and there is no specific treatment for the disease as yet. MicroRNA (miRNA) may play a role in the process of beryllium-induced pulmonary fibrosis. Objective To construct a microRNA-21 (miR-21) interfering cell line, and to investigate the effect of miR-21 on beryllium sulfate (BeSO4)-induced fibrosis in human lung adenocarcinoma alveolar basal epithelial cells (A549 cells) and its potential mechanism. Methods The miR-21 target genes were predicted by the online database miRBase and verified by experiments using dual luciferase reporter gene. After transfecting A549 with miR-21interference lentivirus, puromycin was used to select a stable cell line. An in vitro model of pulmonary fibrosis was established using BeSO4 infecting A549 cells with a concentration of 10 μmol·L−1 and an exposure time of 48 h. Then the treated cells were divided into control group, model group, miR-21 interference group, and miR-21 interference control group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the relative expression level of miR-21 gene. Western blotting was used to detect the relative expression levels of TGF-β1/Smads pathway related proteins [Smad2, Smad3, p-Smad2, p-Smad3, Smad7, and transforming growth factor-β1 (TGF-β1)], myofibrosis cell marker α-smooth muscle actin (α-SMA), andextracellular matrix collagen-I (COL-I) and collagen-Ⅲ (COL-Ⅲ). Results The miRBase predicted that miR-21 had a binding site with Smad7, and the results of the dual luciferase reporter gene experiment showed that the target gene of miR-21 was Smad7. The construction of miR-21 interfered with A549 cell line was successful. Compared with the control group, the relative expression of miR-21 gene in the model group increased by 97.57%; the relative expression of Smad7 protein in the model group decreased by 15.48%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ increased by 13.55%, 35.72%, 18.35%, 35.75%, 25.52%, 31.58%, 24.61%, and 11.66% respectively (P<0.05). Compared with the interference control group, the miR-21 gene expression level in the interference group decreased by 28.96%; the relative expression of Smad7 protein increased by 19.07%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ decreased by 8.01%, 19.95%, 14.56%, 19.37%, 11.95%, 10.96%, 18.81%, and 31.36% repectively (P<0.05). There was no statistically significant difference in the gene abd protein expression levels of each gene between the model group and the interference control group (P>0.05). Conclusion In an in vitro model of pulmonary fibrosis induced by beryllium compounds, miR-21 may promote fibrosis by targeting Smad7 to regulate the TGF-β1/Smad signaling pathway.
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OBJECTIVE: To investigate the effect of beryllium sulfate( BeSO_4) on apoptosis of human embryonic lung fibroblast( MRC-5 cell). METHODS: MRC-5 cells were cultured in vitro and randomly divided into 6 groups: a control group,a low-,medium- and high-dose BeSO_4 group,an antagonist group,and an activator group. The former 4 groups were given final concentrations of 0,1,10 and 100 μmol / L of BeSO_4,respectively. The combined treatment of BeSO_4and2-aminoethoxydiphenyl borate( 10 μmol / L final concentration) was used in the antagonist group. The combined treatment of BeSO_4 and inositol triphosphate( IP3)( 10 μmol / L final concentration) was used in the activator group. After 24 and48 hours of culture,the cells were harvested. The apoptosis of MRC-5 cells was detected by flow cytometry. The intracellular calcium ion( Ca~(2+)) was detected using laser scanning confocal microscope. Quantitative real-time polymerase chain reaction was used to detect the relative expression of IP_3RⅢ and B-cell lymphoma-2( BCL-2) mRNA and the protein expression of IP_3RⅢ and IP3 were detected by enzyme-linked immunosorbent assay. RESULTS: The apoptosis rates of cells in the 3 BeSO_4 dose groups at the time points of 24 and 48 hours were lower than those in the control group at the same time points( P < 0. 05). The apoptosis rate of the antagonist group was lower than those in medium-dose BeSO_4 group and control group at the same time points( P < 0. 05). At the time point of 48 hours,the apoptosis rate of the activator group was lower than that of control group( P < 0. 05) and higher than that of the medium-dose BeSO_4group( P < 0. 05). As for the Ca~(2+)concentration at time point of 24 hours,the low-dose BeSO_4 group was lower than the control group( P < 0. 05),and the high-dose BeSO_4 group was higher than the control group( P < 0. 05). The Ca~(2+)concentrations at time point of 48 hours in the medium- and high-dose BeSO_4 groups were lower than that in the control group( P < 0. 05). Compared with the medium-dose BeSO_4 group and control group at time points of 24 and 48 hours,the Ca~(2+)concentrations in the antagonist group decreased( P < 0. 05),while thoes of the activator group increased( P < 0. 05). The expression of BCL-2and IP_3RⅢmRNA in the 3 BeSO_4 groups,the activator and antagonist group were higher than those of the control group( P <0. 05). The expression of IP3 R Ⅲ protein at the time point of 24 hours in the medium-dose BeSO_4 group,the activator group and the antagonist group were lower than that of control group( P < 0. 05). The expression of IP_3RⅢ protein at the time point of 48 hours,the high-dose BeSO_4 group was lower than the control group( P < 0. 05); the activator and antagonist groups were higher than the medium-dose BeSO_4group( P < 0. 05). The expression of IP3 protein in the lowand medium-dose BeSO_4 groups and the activator group were higher than that in the control group( P < 0. 05). The expression of IP3 protein in activator group was higher than the medium-dose group( P < 0. 05). CONCLUSION: BeSO_4 might change the Ca~(2+)concentration and inhibite the apoptosis of MRC-5 cell through regulating the IP3 R / Ca~(2+)pathway,IP3 can improve the decrease of Ca~(2+)concentration in MRC-5 cells induced by BeSO_4.
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OBJECTIVE: The change of DNA methylation of thymocyte differentiation antigen-1( Thy-1) was observed in beryllium sulfate( Be SO4) stimulated human fetal lung fibroblast( MRC-5 cell) to explore the effects of Thy-1 in Be SO4 induced lung fibrosis. METHODS: MRC-5 cell culture in vitro model was used. The final concentrations of Be SO4were1. 0,10. 0 and 100. 0 μmol / L( low-,medium- and high-dose groups). The control was untreated. Other 2 intervention groups were the 5-azacytidine( AZC) intervention group( 10. 0 μmol / L of AZC and 10. 0 μmol / L Be SO4) and the trichostatin A( TSA) intervention group( 0. 5 μmol / L of TSA and 10. 0 μmol / L Be SO4). The cells were collected 24,48 and 72 hours after exposure. Real-time quantitative polymerase chain reaction( PCR) was used to determine the relative expression of collagen typeⅠ( Col Ⅰ),collagen type Ⅲ( Col Ⅲ),α-smooth muscle actin( α-SMA) and Thy-1 mRNA.The nested landed methylation specific PCR was used to detect the Thy-1 DNA methylation level. RESULTS: At 24 hours,the relative expression level of Col Ⅲ mRNA in MRC-5 cells showed an increasing trend with increasing dose( P < 0. 05);at 48 and 72 hours,the relative expression levels of Col Ⅰ,Col Ⅲ and α-SMA mRNA in MRC-5 cells increased with the increasing dose( P < 0. 05). All these 3 indicators in MRC-5 cells of 3 dose groups increased with the increase of expose time( P < 0. 05). The relative expression level of Thy-1 mRNA in MRC-5 cells of all 3 dose groups were lower than that in control( P < 0. 05). The relative expression level of Thy-1 mRNA of the high-dose group was lower than that of the lowdose group( P < 0. 05). The Thy-1 DNA methylation levels in the medium- and high-dose groups were both higher than that of the control( P < 0. 05). The Thy-1 DNA methylation levels of the 3 dose groups increased with the increasing dose( P < 0. 05). The Thy-1 DNA methylation levels of MRC-5 cells in the 2 intervention groups were higher than that of the control( P < 0. 05),but there was no significant difference when compared with the medium-dose group( P > 0. 05).CONCLUSION: Be SO4 stimulation can induce the fibrosis of MRC-5 cells. In this process,the Thy-1 DNA methylation level increases,while the Thy-1 mRNA expression level decrease. Thy-1 DNA methylation might be one of the important mechanisms of lung fibrosis induced by Be SO4.